Advances in Assisted Reproductive Techniques

This article is from the August 2020 Horse Deals magazine.

For many years, artificial insemination using chilled and frozen semen has been the most widely used assisted reproductive technique (ART) in the horse-breeding industry.

ASSISTED REPRODUCTIVE TECHNIQUES

• AI - Fresh, Chilled and Frozen
• Embryo Transfer
• Ovum Pickup (OPU) - OT, IVF, ICSI & embryo sex determination
• Cloning
• Embryo and Oocyte Freezing

Embryo transfer (ET) in cattle has been well established for well over twenty years as a way to increase the number of progeny per year from a genetically superior animal. The use of embryo transfer in horses has been much slower to develop, partly due to a lack of commercial/economic incentive, combined with a much lower yield of embryos. Cattle can be successfully super-ovulated (given a program of drugs similar to women having IVF), producing up to 20-30 embryos per flush, compared with horses where only one embryo per flush is expected unless the mare has ovulated from both ovaries.

The most relevant and useful application of ET is in the production of offspring from mares still competing, which increases the ability and likelihood of improving the genetics of equine athletes. This seems like an ideal scenario when previously mares were only bred once they were older, retired and less fertile, reducing the chance of producing many offspring. This is not always as easy as it sounds, and sometimes owner expectations exceed what can be achieved in reality. Exercise, heat and stress can disrupt ovarian function while frequent joint injections have been seen to lower the overall fertility of mares and stallions. Furthermore, some disruption to exercise schedule should be expected, although every effort is made to plan breeding around the mare’s work schedule.

The process of ET involves the donor mare being bred with either fresh, chilled or frozen semen; fertilisation takes place within the mare’s fallopian tube and the developing embryo is present in the mare’s uterus by the sixth day following fertilisation. Once the embryo is in the uterus it can be flushed out using special tubing through a cup with a filter; this is usually done on day seven to eight after ovulation, depending on mare age, time of year and type of semen used. The gaps in the filter are smaller than the smallest embryo, therefore the cup will catch and retain the embryo during the flushing process. The contents of the cup are examined under a microscope and the embryo which is approximately the size of a speck of dust is located, washed and held in a special medium until the recipient mare is prepared for transfer. It is ideal to select a couple of recipients for each donor mare flush to increase the chances of perfect synchronicity, and provides us with a choice so we can select the best recipient on the day, and therefore increase the chances of the transfer being successful. Studies show more success with transfer when recipients have ovulated two days after the donor mare. ET is widely available throughout Australia, although a high level of skill is needed to achieve high transfer and pregnancy rates.

Embryos can be frozen using a method called vitrification to be stored indefinitely for the preservation of genetics or until recipients are available for transfer. It involves transferring a small, day six to six and a half ‘morula’ stage embryo through varying concentrations of antifreeze, before submerging it in liquid nitrogen.

Size of the embryo is critical for success, therefore it is important to have a backup plan (i.e. a recipient on standby) if the embryo that is flushed is too big, as this will probably not survive the freezing and thawing processes. A decreased pregnancy rate following thawing and transfer is expected with every frozen embryo, however this can be limited by using an experienced technician.

The next step in breeding technologies, beyond ET, needed to provide many advantages over embryo transfer to warrant the ensuing research needed for development and the extra costs involved in making it commercially viable. Ovum pick-up (OPU) ticks all the boxes!

ADVANTAGES OF OPU/ICSI OVER ET

1. Donor mares can have one-off visits to collection centre when it suits work schedule
2. OPU can be done outside the normal breeding season; in fact, autumn and early spring are often the best times to get a high yield of oocytes
3. Infertility issues in the mare where no embryos are retrieved using conventional ET e.g. inability to ovulate/poor uterine environment/cervical tearing, do not affect the OPU process
4. Embryos can be produced using semen from stallions with low fertility or very limited stocks of semen using ICSI once oocytes are collected using OPU
5. Potentially more embryos can be produced per year (this will hopefully improve as the OPU/ICSI processes improve with practice and research)
6. Embryos are usually frozen after OPU/ICSI and can, therefore, be transferred into recipients when desired; synchronizing with the donor is unnecessary

OPU is where the oocytes (ova or eggs) are aspirated directly from the ovary, either transvaginally or trans-abdominally and identified under a microscope, washed and transferred into a special medium ready for further processing.

Trans-vaginal OPU is the most commonly used technique now; a long rigid ultrasound probe, held within the vagina against the ovary, acts as a visual guide for an attached needle. Once each follicle is visualised, the needle is advanced and the follicle is repeatedly flushed with a solution using a pump. 15-20 follicles may be flushed from the two ovaries during a single OPU session, yielding anything from 0-20 oocytes. A large dominant follicle which is close to ovulation yields a mature oocyte. As previously mentioned and similarly to ET, only one to two mature oocytes are available per flush if only dominant follicles are flushed. However, many immature oocytes can be harvested from multiple smaller follicles. Furthermore, immature follicles are much more stable and easier to transport for further processing. A donor mare in the transitional phase in early spring and late autumn has the ideal ovaries for harvesting as many oocytes as possible each flush. OPU can be carried out every two to three weeks.

Oocyte transfer (OT) has been used in the place of ET for mares with reproductive issues which results in no embryo production. It requires recovery of a mature oocyte using OPU from the donor mare. The recipient mare is synchronised with the donor, her pre-ovulatory follicle is flushed to remove her mature oocyte and the donor mare oocyte is placed in the fallopian tube of the recipient through a flank incision. The recipient is inseminated and the fertilisation of the donor oocyte takes place within the recipient’s fallopian tube. OT has not taken off over the years as it holds only a small advantage over ET and is more invasive for the recipients involved.

Ten to fifteen years ago, much of the research effort was concentrated on IVF, the most widely used ART in humans. To this day only a couple of foals have been born using this technique. It requires the sperm to ‘swim up’ to the ovum in a petri dish for fertilisation to take place. The equine sperm is usually unable to fertilise the ovum in these conditions.

Intra-Cytoplasmic Sperm Injection

1. Immature oocytes matured in an incubator
2. One sperm selected and injected into the cytoplasm of each oocyte using micromanipulator
**3. Fertilisation in vitro (in a petri dish)
4. Incubation/culture for six to eight days - result = embryo!
5. Embryo transferred or frozen

It has become very evident at recent International ART conferences that all research has shifted towards OPU-ICSI as the way forward for the production of embryos. An overwhelming amount of development has taken place in these areas over the last six to eight years, and more equine reproductive clinics and labs are working towards providing these techniques commercially around the world.

Intra-cytoplasmic sperm injection (ICSI) is a really exciting development in equine breeding, but requires expensive equipment in a purpose-built lab run by highly skilled individuals that have dedicated most of their working life to the processes involved. However, it is possible for OPU, which requires less skill than ICSI and less equipment, to become more widely available in Australia soon. Under current importation laws we are unable to import embryos, but hopefully this will change sometime in the future, making it available commercially.

It’s really important for the Australian equine breeding industry to keep up with the new developments in OPU/ICSI to enable us to become more competitive internationally. We don’t always want to be relying on having to import performance horses; we should strive to breed our own quality horses.

CLONING

1. Cells taken from donor animal to be cloned - can be a gelding
2. Unfertilised oocyte from a random mare with DNA material removed, therefore not transferring genetic influence to embryo produced
3. Cell nucleus from donor implanted into oocyte and an electric pulse results in fusion and the start of cell division
4. Resulting embryo transferred into the recipient as normal
5. Cost around £100,000 (approx $180,000 AUD)

A very recent development which was reported in 2014 by Dr C Herrera at Zurich University is sex determination using material extracted from the embryo. Embryos produced by ICSI are biopsied and the genetic material undergo genetic analysis using PCR (polymerase chain reaction). More research in this area may lead to improving our selection of individuals for phenotype, which is already commonplace in the cattle world. Interestingly the collapse of the embryo following biopsy also renders it more suitable for freezing at the blastocyst stage.

Somatic cell nuclear transfer (SCNT), or cloning, is the production of a population of genetically identical individuals. Dolly, the first animal clone was born in 1996, and the first horse clone born in 2003. Since then, Crestview Genetics in the States has produced over 200 horses since 2009. Some highly respected reproductive specialists are concerned that if we rely too heavily on cloning we may not be developing and improving the genetics of our equine athletes, a sideways step so to speak? Since 2012, clones have been allowed to compete FEI, so no matter our personal view, cloning is here to stay.

Article by Dr Noelle Baxter B.V.Sc Cert EM(Stud Med) MRCVS of Sydney Equine Practice

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